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Proteomic analyses identify a novel role for EZH2 in the initiation of cancer cell drug tolerance

ABSTRACT: Acquisition of drug resistance remains a chief impediment to successful cancer therapy, and we previously described a transient drug-tolerant cancer cell population (DTPs) whose survival is in part dependent on the activities of the histone methyltransferases G9a/EHMT2 and EZH2, the latter being the catalytic component of the polycomb repressive complex 2 (PRC2). Here, we applied multiple proteomic techniques to better understand the role of these HMTs in the establishment of the DTP state. Proteome-wide comparisons of lysine methylation patterns revealed that DTPs have increased methylation on K116 of PRC member Jarid2, an event that helps stabilize and recruit PRC2 to chromatin. We also found that EZH2, in addition to methylating histone H3K27, also methylates G9a at K185, and that methylated G9a better recruits repressive complexes to chromatin; similar to complexes recruited by histone H3 methylated at K9. Finally, a detailed histone posttranslational modification (PTM) analysis shows that EZH2, either directly or through its ability to methylate G9a, alters H3K9 methylation in the specific context of H3 serine10 phosphorylation, and primarily in a cancer cell subpopulation that serves as DTP precursors. We also show that combinations of histone PTMs recruit a different set of complexes to chromatin, shedding light on the temporal mechanisms that contribute to drug tolerance.

INSTRUMENT(S): Orbitrap Fusion, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: David Arnott

PROVIDER: MSV000084838 | MassIVE | Fri Jan 24 12:25:00 GMT 2020

REPOSITORIES: MassIVE

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Project description:Chromatin structure and function is maintained by dynamic protein-protein and protein-nucleic acid interactions. Histones are a family of proteins that are abundant chromatin constituents and that carry numerous post-translational modifications (PTMs). Histone PTMs mediate a variety of biological activities, including recruitment of enzymatic readers, writers and erasers that modulate protein activities, DNA replication, transcription and repair. Individual histone molecules contain multiple co-existing PTMs some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. We here present an integrated experimental and computational approach for systems level molecular characterization of PTMs and PTM crosstalk. Using wildtype and engineered mouse embryonic stem cells with perturbations in the Polycomb Repressive Complex 1 (PRC1, suz12-/-), PRC2 (Ring1A/b-/-) and DNA methyltransferases (Dnmt1/3a/3b-/-) we performed comprehensive PTM analysis of histone H3 tails. We identified unique histone H3 PTM features of each of the four cell lines and we detected common combinatorial PTM features across cell lines. Using quantitative middle-down proteomics combined with probabilistic and statistical data analysis we extracted histone H3 PTM profiles for all four mESC systems. PTM crosstalk emerged as mutually exclusive histone PTMs or coordinately regulated PTMs independent of histone peptide abundance in the four model systems. We detected positive crosstalk between adjacent mono-methylated marks but strong negative crosstalk among most of the seven characterized di- and tri-methylations on lysines. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites in histone H3, including H3R2me, H3R8me and H3K37me, which exhibited specific PTM codes suggesting a particular role in chromatin. All histone H3 PTM data is available in our publicly available CrossTalkDB repository at http://crosstalkdb.bmb.sdu.dk

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Proteomic analyses identify a novel role for EZH2 in the initiation of cancer cell drug tolerance

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